Light-induced protein-matrix uncoupling and protein relaxation in dry samples of trehalose-coated MbCO at room temperature

In humid samples of trehalose-coated carboxy-myoglobin (MbCO), thermally driven conformational relaxation takes place after photodissociation of the carbon monoxide (CO) molecule at room temperature. In such samples, because of the extreme viscosity of the external matrix, photodissociated CO cannot diffuse out of the protein and explores the whole (proximal and distal side) heme pocket, experiencing averaged protein heme pocket structures, as a results of the presence of Brownian motions. At variance, in very dry samples, a lower portion of the photodissociated CO diffuses from the distal to the proximal heme pocket side probing in nonaveraged structures. We revisit here the flash photolysis data by Librizzi et al. (2002) and report on new, room temperature experiments in MbCO-trehalose samples, shortly illuminated prior the laser pulse. In dry samples, pre-illumination increased the diffusion of CO from the distal to the proximal heme pocket side, which resulted in less structure than in non-pre-illuminated samples. Such an effect, which is absent in humid samples, stems from a decoupling of the protein internal degrees of freedom from those of the external water-sugar matrix. We suggest that such a decoupling can be brought about by the continuous attempts performed by the protein during pre-illumination to undergo relaxation toward the photodissociated deoxy state. This, in turn, causes a collapse in the hydrogen bond network, which connects the protein surface to the water-sugar matrix, as reported by Cottone et al. (2002) and Giuffrida et al. (2003). In the conclusion section, we discuss the possible involvement of the processes invoked to rationalize the present data, in the function of macromolecules and interactions in living cells.     

The helical repeat of DNA at high temperature.

The increasing number of studies on thermophilic organisms addressed the question of DNA double helix parameters at high temperature. The present study shows that the helix rotation angle per base pair omega of an unconstrained DNA decreases linearly upon temperature increase, up to the premelting range. In the ionic conditions tested, this rule extends to temperatures up to 85 degrees C, which is a common growth temperature for many hyperthermophilic organisms. In addition, the torsional constant K of DNA decreases with temperature, indicating that the energy required to modify the DNA twist is lower at high temperature. These findings have several implications for people working on the structure and enzymology of DNA at high temperature.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic adaptation of prokaryotic organisms at high temperature

One of the central issues of evolutionary genomics is to find out the adaptive strategies of microorganisms to stabilize nucleic acid molecules under high temperature. Thermal adaptation hypothesis gives a link between G+C content and growth temperature if there is a considerable variation of guanine and cytosine content between species. However, there has been a long-standing debate regarding the correlations between genomic GC content and optimal growth temperature (Topt). We urged that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting: (i) nucleic acid thermostability and (ii) stability of codon-anticodon interactions. Moreover, in Bacillaceae family we have demonstrated that a higher genomic GC level do not have any role in stabilizing mRNA secondary structure at high growth temperature. Comparative analysis between homologous sequences of thermophilic Thermus thermophilus and mesophilic Deinococcus radiodurans suggests that increased levels of GC contents in the coding sequence corresponding to strand structure of Thermus thermophilus genes have stabilizing effect on the mRNA secondary structure, whereas increased levels of GC contents in coding sequences corresponding to aperiodic structure have destabilizing effect on the mRNA secondary structure. In this perspective, a critical review of thermal adaptation hypothesis is further advocated.     

Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers

X-ray structures of carbonmonoxymyoglobin (MbCO) are available for different pH values. We used conventional electrostatic continuum methods to calculate the titration behavior of MbCO in the pH range from 3 to 7. For our calculations, we considered five different x-ray structures determined at pH values of 4, 5, and 6. We developed a Monte Carlo method to sample protonation states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and the titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carlo method. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas the x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreement with experimental results compared to calculations using only a single conformation. The most striking feature of pH-dependent conformational changes in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as proposed earlier.     

Defect in expression of heat-shock proteins at high temperature in xthA mutants.

Escherichia coli mutants lacking exonuclease III (xthA) are defective in the induction of heat-shock proteins upon severe heat-shock treatment (upshift from 30 to 50 degrees C) but not mild heat-shock treatment (upshift from 30 to 42 degrees C). We show that this defect is due to the xthA mutation by complementation. Furthermore, increasing the gene dosage of xthA+ prolongs the synthesis of heat shock proteins seen after a shift to 42 degrees C. Increasing the gene dosage of htpR+ partially suppresses the defect of xthA mutants in the synthesis of heat-shock proteins at 50 degrees C. When an xthA strain was incubated at 42 degrees C before a shift to 50 degrees C, it was then able to carry out the synthesis of heat-shock proteins at 50 degrees C.     

Inhibition of expression of tomato-ripening genes at high temperature

Abstract. Ripening tomato fruits incubated at 35°C fail to achieve normal pigmentation, soften little and show a marked decline in ethylene evolution. Labelling studies in vivo indicate that protein synthesis continues throughout incubation at 35°C although the spectrum of labelled proteins is different to that observed at 25°C. Translation of mRNAs in vitro shows traces of several 'heat-shock' mRNAs at 35°C and the loss of several others normally found in fruit ripened at 25°C. Using ripening-related cDNA clones as hybridization probes the expression of 12 ripening-related genes was followed during incubation at 25°C and 35°C. In general, there was a marked decline in the amounts of these mRNAs following incubation of ripening fruit at 35°C. In particular, mRNA homologous to pTOM 6, a cDNA clone coding for polygalacturonase, a major cell wall degrading enzyme, showed a rapid decline following incubation at 35°C and after 72-h at elevated temperature was undetectable. There was no recovery of expression during 120 h at 35°C and the application of exogenous ethylene did not overcome the inhibition of ripening or lead to the renewed accumulation of polygalacturonase mRNA. It is proposed that the failure to soften normally at elevated temperature is due, in part, to the suppression of polygalacturonase mRNA and that the inhibition of other facets of ripening at 35°C is due to the inhibition or reduced expression of other, as yet unidentified, ripening-related genes.     

Molecular dynamics simulation of sucrose- and trehalose-coated carboxy-myoglobin

We performed a room temperature molecular dynamics (MD) simulation on a system containing 1 carboxy-myoglobin (MbCO) molecule in a sucrose-water matrix of identical composition (89% [sucrose/(sucrose + water)] w/w) as for a previous trehalose-water-MbCO simulation (Cottone et al., Biophys J 2001;80:931-938). Results show that, as for trehalose, the amplitude of protein atomic mean-square fluctuations, on the nanosecond timescale, is reduced with respect to aqueous solutions also in sucrose. A detailed comparison as a function of residue number evidences mobility differences along the protein backbone, which can be related to a different efficacy in bioprotection. Different heme pocket structures are observed in the 2 systems. The joint distribution of the magnitude of the electric field at the CO oxygen atom and of the angle between the field and the CO unit vector shows a secondary maximum in sucrose, absent in trehalose. This can explain the CO stretching band profile (A substates distribution) differences evidenced by infrared spectroscopy in sucrose- and trehalose-coated MbCO (Giuffrida et al., J Phys Chem B 2004;108:15415-15421), and in particular the appearance of a further substate in sucrose. Analysis of hydrogen bonds at the protein-solvent interface shows that the fraction of water molecules shared between the protein and the sugar is lower in sucrose than in trehalose, in spite of a larger number of water molecules bound to the protein in the former system, thus indicating a lower protein-matrix coupling, as recently observed by Fourier transform infrared (FTIR) experiments (Giuffrida et al., J Phys Chem B 2004;108:15415-15421).     

Effect of hypercapnia on thermoregulation at high ambient temperature

The reported investigations were carried out on rabbits exposed for three hours to ambient temperature of 25 degrees C or 35 degrees breathing athmospheric air (controls) or gas mixtures containing 4% or 7% of CO2. During the exposure to 35 degrees C in rabbits breathing the gas mixture with 7% of CO2 the rise of rectal temperature was significantly greater, heat elimination from the auricular surface was increased, whereas the oxygen uptake was increased insignificantly. In tracheostomized rabbits breathing the gas mixture with 7% of CO2 at 32 degrees C the respiratory rate decreased but the respiration volume increased as compared with the animals breathing atmospheric air. It seems that the hyperthermic effect of hypercapnia demonstrated in this work can be attributed to the impairment of heat elimination through the upper airways due to an inhibition of thermal panting.     

Effects of air movement on swine at high temperature

Sixty pigs were used to study the effects of air movement (0.05, 0.5, and 1.0 m/s) at constant air temperatures of either 5.6 or 11.1°C above optimum, and at 3 different weight ranges (36–50kg, 54–68 kg, and 73–86 kg) during the growing-finishing stages. An air velocity of 0.5 m/s produced significantly greater weight gain and feed conversion than did 0.05 m/s. An air velocity of 1.0 m/s produced intermediate results.     

DNA cleavage and religation by human topoisomerase II alpha at high temperature.

A common DNA religation assay for topoisomerase II takes advantage of the fact that the enzyme can rejoin cleaved nucleic acids but cannot mediate DNA scission at suboptimal temperatures (either high or low). Although temperature-induced DNA religation assays have provided valuable mechanistic information for several type II enzymes, high-temperature shifts have not been examined for human topoisomerase IIalpha. Therefore, the effects of temperature on the DNA cleavage/religation activity of the enzyme were characterized. Human topoisomerase IIalpha undergoes two distinct transitions at high temperatures. The first transition occurs between 45 and 55 degrees C and is accompanied by a 6-fold increase in the level of DNA cleavage at 60 degrees C. It also leads to a loss of DNA strand passage activity, due primarily to an inability of ATP to convert the enzyme to a protein clamp. The enzyme alterations that accompany the first transition appear to be stable and do not revert at lower temperature. The second transition in human topoisomerase IIalpha occurs between 65 and 70 degrees C and correlates with a precipitous drop in the level of DNA scission. At 75 degrees C, cleavage falls well below amounts seen at 37 degrees C. This loss of DNA scission appears to result from a decrease in the forward rate of DNA cleavage rather than an increase in the religation rate. Finally, similar high-temperature alterations were observed for yeast topoisomerase II and human topoisomerase IIbeta, suggesting that parallel heat-induced transitions may be widespread among type II topoisomerases.     

Thermal behavior of bovine β-trypsin at physiological temperature range

The effects of temperature on the steady-state kinetics of β-trypsin hydrolysis of α-Ntosyl-l-arginine methyl ester (k_cat, K_m) and its inhibition by phenylguanidinium ion (K_i) were studied in the temperature range 27–37 °C, at 1 °C intervals, pH 8.0. Within this temperature range inhibition of β-trypsin by phenylguanidine was strictly competitive. The Eyring and van't Hoff plots were nonlinear; interpretation of the data was based on two possible alternatives: in the first, there occurs a thermal transition centered at 31 °C, characterized by ΔH° = 42.2 ± 8.7 kcal/mol and ΔS ° = 138 ± 29 e.u. According to the second interpretation the phenomenon would be determined by a large value of Δ Cp; its value was estimated to be ΔCp = ?7192 cal/deg · mol. A decision as to what interpretation is more adequate must wait until further experimental information is obtained.     

The temperature sensitive mutant 72c. II. Accumulation at high temperature of ppGpp and pppGpp in the presence of protein synthesis.

A heat sensitive mutant of E. coli has been analyzed. A shift to restrictive temperature leads to an accumulation of ppGpp and pppGpp in both the parental and the mutant strains (both are relA+). The pool of these compounds is shown to decrease with time after the temperature shift in the case of the parental strain, but remains at the same elevated level in the case of the mutant. The temperature shift of the mutant leads to an apparent reduction of stable RNA synthesis; this inhibition can be released by chloroamphenicol or tetracycline. Gross protein synthesis is more or less unaffected at restrictive temperature. In the parental strain little effect is seen on RNA and protein synthesis after the temperature shift. A relA derivative of the mutant does not show the same inhibition of RNA synthesis at high temperature. Sedimentation analysis suggests that mutant 70S ribosome are more stable, when exposed to a lowered Mg2+ concentration, than are 70S ribosomes from the parental strain. In addition, the relative amounts of the two forms of ribosomal protein S6, which can be obtained on DEAE chromatography (Held et al., 1973), are significantly changed in the mutant.